Inflammation is a complex of sequential changes expressing the response to damage of cells and vascularized tissues. When tissue injury occurs, whether it be caused by bacteria, trauma, chemicals, heat, or any other phenomenon, the substance histamine, along with other humoral substances, is liberated by the damaged tissue into the surrounding fluids. It is a protective attempt by the organism to remove the injurious stimuli as well as initiating the healing process.
The main features of the inflammatory response are vasodilation, i.e. widening of the blood vessels to increase the blood flow to the infected area; increased vascular permeability which allows diffusible components to enter the site; cellular infiltration by chemotaxis; or the directed movement of inflammatory cells through the walls of blood vessels into the site of injury; changes in biosynthetic, metabolic, and catabolic profiles of many organs; and activation of cells of the immune system as well as of complex enzymatic systems of blood plasma. Inflammation which runs unchecked can, however, lead to a host of diseases, such as hay fever, atherosclerosis and rheumatoid arthritis.
There are two forms of inflammation, commonly referred to as acute inflammation and chronic inflammation. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. Acute inflammation can be divided into several phases. The earliest, gross event of an inflammatory response is temporary vasoconstriction, i.e., narrowing of blood vessels caused by contraction of smooth muscle in the vessel walls which can be seen as blanching (whitening) of the skin. This is followed by several phases that occur minutes, hours and days later. The first is the acute vascular response which follows within seconds of the tissue injury and lasts for several minutes. This results from vasodilation and increased capillary permeability due to alterations in the vascular endothelium which leads to increased blood flow (hyperemia) that causes redness (erythema) and the entry of fluid into the tissues (edema).
The acute vascular response can be followed by an acute cellular response which takes place over the next few hours. The hallmark of this phase is the appearance of granulocytes, particularly neutrophils, in the tissues. These cells first attach themselves to the endothelial cells within the blood vessels (margination) and then cross into the surrounding tissue (diapedesis). During this phase erythrocytes may also leak into the tissues and a hemorrhage can occur. If the vessel is damaged, fibrinogen and fibronectin are deposited at the site of injury, platelets aggregate and become activated, and the red cells stack together in what are called “rouleau” to help stop bleeding and aid clot formation. The dead and dying cells contribute to pus formation. If the damage is sufficiently severe, a chronic cellular response may follow over the next few days. A characteristic of this phase of inflammation is the appearance of a mononuclear cell infiltrate composed of macrophages and lymphocytes. The macrophages are involved in microbial killing, in clearing up cellular and tissue debris, and in remodeling of tissues.
Chronic inflammation is an inflammatory response of prolonged duration—weeks, months or indefinitely—whose extended time course is provoked by persistence of the causative stimulus to inflammation in the tissue. The inflammatory process inevitably causes tissue damage and is accompanied by simultaneous attempts at healing and repair. The exact nature, extent and time course of chronic inflammation is variable depending on a balance between the causative agent and the attempts of the body to remove it.
Etiological agents producing chronic inflammation include:
(i) infectious organisms that can avoid or resist host defenses and so persist in the tissue for a prolonged period, including Mycobacterium tuberculosis, Actinomycetes, and numerous fungi, protozoa and metazoal parasites. Such organisms are in general able to avoid phagocytosis or survive within phagocytic cells, and tend not to produce toxins causing acute tissue damage;
(ii) infectious organisms that are not innately resistant but persist in damaged regions where they are protected from host defenses. An example is bacteria which grow in the pus within an un-drained abscess cavity where they are protected both from host immunity and from blood-borne therapeutic agents, e.g. antibiotics. Some locations are particularly prone to chronic abscess formation, e.g. bone and pleural cavities;
(iii) irritant non-living foreign material that cannot be removed by enzymatic breakdown or phagocytosis. Examples include a wide range of materials implanted into wounds (wood splinters, grit, metals and plastics), inhaled (silica dust and other particles or fibers) or deliberately introduced (surgical prostheses, sutures, etc.), including transplants. Dead tissue components that cannot be broken down may have similar effects, e.g. keratin squames from a ruptured epidermoid cyst or fragments of dead bone (sequestrum) in osteomyelitis.
(iv) in some cases the stimulus to chronic inflammation may be a normal tissue component. This occurs in inflammatory diseases where the disease process is initiated and maintained because of an abnormality in the regulation of the body's immune response to its own tissues—the so-called auto-immune diseases. This response is seen in elderly and aging subjects; and
(v) for many diseases characterized by a chronic inflammatory pathological process the underlying cause remains unknown. An example is Crohn's disease.
Although the production of pro-inflammatory cytokines by cells of the innate immune system plays an important role in mediating the initial host defense against invading pathogens (O'Neill, L. A. et al., Immunol. Today, (2000), 21 (5):206-9), an inability to regulate the nature or duration of the host's inflammatory response can often mediate detrimental host effects as observed in chronic inflammatory diseases. Additionally, in the early stages of sepsis, the host's inflammatory response is believed to be in a hyperactive state with a predominant increase in the production of pro-inflammatory cytokines that mediate host tissue injury and lethal shock (Cohen, J., Nature, (2002), 420 (6917):885-91). In this regard, the ability to suppress pro-inflammatory cytokines and/or enhance anti-inflammatory cytokines, i.e. IL-10, has been shown to severely reduce the toxic effects of endotoxin (Berg, D. J. et al., J. Clin. Invest., (1995), 96 (5):2339-47; and Howard, M. et al., J. Exp. Med., (1993), 177 (4):1205-8).
Inflammatory cytokines released by immune cells have been shown to act on the central nervous system (CNS) to control food intake and energy homeostasis (Hart, B. L., Neurosci. Biobehav. Rev., (1988), 12 (2):123-37). Decrease in food intake or anorexia is one of the most common symptoms of illness, injury or inflammation (Kotler, D. P., Ann. Intern. Med., (2000), 133 (8):622-34). Cytokines, such as IL-1β, IL-6 and TNF-α, have been implicated in wasting associated with inflammation (Ershler, W. B. et al., Annu. Rev. Med., (2000), 51:245-70), chronic low-grade inflammation in aging (Bruunsgaard, H. et al., Curr. Opin. Hematol., (2001), 8 (3):131-6) and atherosclerosis (Bochkov, V. N. et al., Nature, (2002), 419 (6902):77-81).
What is need in the art is the regulation if inflammatory cytokine production by endogenous factors such as ghrelin analogues to ameliorate a wide variety of ailments and disease conditions.
Human ghrelin, an orexigenic hormone, is synthesized as a preprohormone and proteolytically processed to yield a 28-amino acid peptide of the following sequence: H-Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-NH2 (SEQ ID: 1) (Kojima, M. et al., Nature, (1999), 402 (6762):656-60). Ghrelin is produced predominantly by epithelial cells lining the fundus of the stomach, however, smaller amounts are produced in the placenta, kidney, pituitary and hypothalamus. A core region present in ghrelin, responsible for observed activity, comprises the four N-terminal amino acids wherein the serine in the third position is normally modified with n-octanoic acid. In addition to acylation by n-octanoic acid, native ghrelin may also be acylated with n-decanoic acid (Kaiya, H. et al., J. of Biol. Chem., (2001), 276 (44):40441-8).
The ghrelin receptor was known well before the peptide was discovered. Cells within the anterior pituitary gland bear a receptor that, when activated, powerfully stimulates GH secretion, mainly at the hypothalamic level (Kojima, M. et al., Nature, (1999), 402 (6762):656-60). That receptor was named the growth hormone secretagogue receptor (“GHS-R”) (Ukkola, O. and Pöykkö, S., 2002 Ann. Med., (2002), 34 (2):102-8; and Kojima, M. et al., Nature, (1999), 402 (6762):656-60). It is postulated that ghrelin enhances the activity of growth hormone releasing hormone (GHRH)-secreting neurons and, concomitantly, acting as a functional somatostatin antagonist (Ghigo, E. et al., Euro. J. Endocrinol., (1997), 136 (5):445-60).
The GHS-R and its subtypes are not restricted to the hypothalamus-pituitary unit, but are present in other central and peripheral tissues, such as heart and adipose tissues (Papotti, M. et al., J. Clin. Endocrinol. Metab., (2000), 85 (10):3803-7). GHS-R is also expressed in the pancreas (Guan, X. M. et al., Brain Res., (1997), 48 (1)-23-9; and Volante, M. et al., J. Clin. Endocrinol. Metab., (2002), 87 (3):1300-8). The physiological actions of ghrelin, as well as those of synthetic GHS, are not restricted to GH secretion. Ghrelin has been shown to stimulate lactotroph and corticotroph hormone secretion has orexigenic and cardiovascular actions, shows anti-proliferative effects on thyroid and breast tumors, as well as regulating gastric motility and acid secretions through vagal mediation (Ukkola, O. and Pöykkö, S., Ann. Med., (2000), 34 (2):102-8). Most importantly, expression of the GH and GH secretagogue receptors and ghrelin has been detected in all immune cells, including human T and β cells as well as neutrophils (Hattori, N. et al., J. Clin. Endocrinol. Metab., (2001), 86 (9):4284-91).
Ghrelin is a physiological ligand for the growth hormone secretagogue receptor (GHS-R) and as such, powerfully stimulates secretion of growth hormone. Ghrelin acts by increasing intracellular Ca2+ concentration. The ghrelin signal is integrated with that of growth hormone releasing hormone and somatostatin to control the timing and magnitude of growth hormone secretion. In both humans and rodents, ghrelin functions to increase hunger through its action on hypothalamic feeding centers (Cummings, D. E. et al., Diabetes, (2001), 50 (8):1714-9). Ghrelin also functions in energy metabolism and gastric acid secretion and motility (Date, Y. et al., Diabetes, (2002), 51 (1):124-9). Ghrelin has been found to have a variety of positive effects in cardiovascular function, such as increased cardiac output, however, it is not totally clear whether the cardiovascular effects are a directed effect of ghrelin or represent an indirect effect of ghrelin's ability to stimulate growth hormone secretion. In addition, the wide tissue distribution of GHS-R in the lymphoid system suggests that ghrelin and GHS-R ligands can function as signal modulators between the endocrine, nervous and immune systems.
Ghrelin, via functional cell surface GHS-R, exerts both specific and selective inhibitory effects on the expression and production of inflammatory cytokines such as IL-1β, IL-6 and TNF-a, by human PBMCs and T cells. The GHS-R on primary and cultured human T cells, similar to other classical GPCRs, elicits a potent intracellular calcium release upon ligation with its natural ligand, ghrelin, and is preferentially associated with GM1 lipid rafts upon cellular activation. Consistent with expression of functional GHS-R on T cells, ghrelin actively induces actin polymerization within T cells. Similar to chemokines (SDF-1), ghrelin treatment led to the cellular polarization of leukocytes and actin distribution changes from a linear cortical pattern in resting lymphocytes to more concentrated patterns at the leading edge and contact zones in polarized and activated T cells (Taub, D. D. et al. Science, (1993), 260 (5106):355-8; and Inui, A., Cancer Res., (1999), 59 (18):4493-501). These GPCR-like redistribution patterns show an important role for GHS-R in immune cell signaling and trafficking.
Through a number of analytical techniques, it has been demonstrated that ghrelin is endogenously produced and secreted by both T cells and PBMCs in a fashion similar to many immune-derived cytokines. The majority of T cells examined from human donors were found to constitutively express low levels of endogenous ghrelin, which is significantly increased upon cellular activation. Activated T cells express and secrete the ghrelin protein, exhibiting that pre-pro peptide must be actively cleaved in T cells to yield the active ghrelin peptide. Similar to several cytokines (e.g., TGF-p) and hormones (e.g., TSH), these precursor proteins are synthesized and subsequently stored for immediate cleavage and use when needed. Furthermore, the expression and secretion of a mature form of ghrelin from T cells post activation via T cell receptor ligation has been demonstrated. Given that gastrectomy results in only a 35 to 50% decline in circulating ghrelin and that ghrelin levels increase to two thirds of pre-gastrectomy levels in human subjects, it has been shown that other tissues compensate for maintaining the circulating ghrelin (Hosoda, H. et al., J. Biol. Chem., (2003), 278 (1):64-70). Secretion of ghrelin from T cells shows that immune cell-derived ghrelin makes up part of the residual concentration of circulating ghrelin. In addition, ghrelin is also regarded as the only known hormone where the hydroxyl group of its third serine residue is acylated by n-octanoic acid and this acylation is critical for some of the biological activities of this polypeptide (Kojima, M. et al., Nature, (1999), 402 (6762):656-60). N-terminal acylated peptides are known to preferentially aggregate in cholesterol rich micro-domains (Basa, N. R. et al., Neurosci. Lett., (2003), 343 (1):25-8), and ghrelin is immunoreactive in activated T cells and is highly co-localized within cholesterol-rich GM1+ domains. These results show that ghrelin is selectively targeted to the plasma membrane to facilitate interaction with its own transmembrane receptor to optimally mediate receptor-ligand interactions. Such a pathway shows the role of ghrelin in the control of immune responses. In addition, localized production of ghrelin plays a critical role in the immediate control of ongoing and leptin-mediated responses within the local microenvironment.
Ghrelin has been effective in treating inflammation in a mammalian subject (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]). In particular, the inflammation can be associated with a viral, bacterial, parasitic or fungal infection. Viral infections treatable with ghrelin may include Herpes simplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1 and Human Immunodeficiency virus type-2. (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Bacterial infections that cause inflammation that can be treated with ghrelin (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]) include M. tuberculosis, M bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsia species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica and other Yersinia species (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Inflammation treatable with ghrelin (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]) can also be caused by parasites including Toxoplasma gondii, Plasmodium, Trypanosoma brucei, Trypanosoma cruzi, Leishmania, Schistosoma and Entamoeba histolytica or fungi such as Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi and Alternaria alternate (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Inflammation caused by liver toxicity or transplant rejection is also treatable by ghrelin (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]). The liver toxicity may be associated with cancer therapy. In some instances, the cancer therapy, such as chemotherapy, may bring about liver toxicity. Liver toxicity brought about by both chemotherapy and apoptosis may be treatable by administration of ghrelin, ghrelin agonists or ghrelin antagonists (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Inflammation associated with cancer is also treatable with ghrelin (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]). Such cancers include lymphoma, leukemia, mycosis fungoide, carcinoma, adenocarcinoma, sarcoma, glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or AIDS-related sarcoma, metastatic cancer, bladder cancer, brain cancer, nervous system cancer, glioblastoma, ovarian cancer (International Patent Application No. PCT/AU02/00582 [WO 02/090387]; and Gaytan, F. et al., J. Clin. Endocri. Metab., (2005), 90 (3):1798-804), skin cancer, liver cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer (International Patent Application No. PCT/AU02/00582 [WO 02/090387]), breast cancer (International Patent Application No. PCT/AU02/00582 [WO 02/090387]; and Cassoni, P. et al., J. Clin. Endocri. Metab., (2001), 86 (4):1738-45), epithelial cancer, renal cancer (Jungwirth, A. et al., Proc. Natl. Acad. Sci. U.S.A., (1997), 94 (11):5810-3), genitourinary cancer, pulmonary cancer (Ghé, C. et al., Endocrinology, (2002), 143 (2):484-91), esophageal carcinoma (Nwokolo, C. U. et al., Gut, (2003), 52 (5):637-40), head and neck carcinoma (Jozkow, P. et al., Head Neck, (2005), 27 (3):243-7), hematopoietic cancer, testicular cancer (Gaytan, F. et al., J. Clin. Endocri Metab., (2004), 89 (1):400-9), colo-rectal cancer (Dagnaes-Hansen, H. et al., Anticancer Res., (2004), 24 (6):3735), prostatic cancer (Jeffery, P. L. et al., Endocrinology, (2002), 172:R7-11), and pancreatic cancer (Volante, M. et al., J. Clin. Endocri. Metab., (2002), 87 (3):1300-8); and International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Ghrelin has been shown to treat inflammatory diseases (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]) such as asthma, reactive arthritis (Granado, M. et al., Am. J. Physiol. Endocrinol. Metab., (2005), 288 (3):E-486-92), hepatitis (Wallace, J. D. et al., J. Clin. Endocri. Metab., (2002), 87 (6):2751-9), spondyarthritis, Sjogren's syndrome, Alzheimer's disease (U.S. Pat. Nos. 6,686,359 and 6,566,337; and Obermayr, R. P. et al., Gerontology, (2003), 49 (3):191-5), and atopic dermatitis or inflammatory diseases associated with an autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis (Otero, M. et al., Rheumatology (Oxford), (2004), 43 (3):306-10), systemic vasculitis, insulin dependent diabetes mellitus (Nieves-Riviera, F. et al., Growth Regul., (1993), 3:235-44), multiple sclerosis and muscular dystrophy (U.S. Patent Publication No. 2003/0139348), experimental allergic encephalomyelitis (Ikushima, H. et al., J. Immunol., (2003), 171:2769-72), psoriasis (Edmondson, S. R. et al., Endocri. Rev., (2003), 24 (6):737-64), Crohn's disease (Slonim, A. E. et al., N. Engl. J. Med., (2000), 342 (22):1633-7), inflammatory bowel disease (Chen, K. et al., Surgery, (1997), 121 (2):212-8), ulcerative colitis, Addison's disease (Arvat, E. et al., Neuroendocrinology, (1999), 70 (3):200-6), alopecia aretea, celiac disease (Peracchi, M. et al., Am. J. Gastroenterol., (2003), 98 (11):2474-8; and Capristo, E. et al., Scand. J. Gastroenterol., (2005), 40 (4):430-6), thyroid disease (Riis, A. L. et al., J. Clin. Endocrin. Metab., (2003), 88 (2):853-7), and scleroderma. Inflammation as a result of a burn may also benefit from treatment with ghrelin as may inflammation of the lung (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]). Inflammation may also cause a subject to lose appetite, particularly when the inflammation is low grade and/or in an aging subject (International Patent Application No. PCT/US2005/016565 [WO 2005/110463]).
Inflammatory cytokines released by immune cells have been shown to act on the central nervous system (CNS) to control food intake and energy homeostasis (Hart, B. L., Neurosci. Biobehay. Rev., (1988), 12 (2):123-37). Decrease in food intake or anorexia is one of the most common symptoms of illness, injury or inflammation (Kotler, D. P., Ann. Internal Med., (2000), 133 (8):622-34). Cytokines such as IL-1β, IL-6 and TNF-α have been implicated in wasting associated with inflammation (Ershler, W. B. and Keller, E. T., Annu. Rev. Med., (2000), 51:245-70), chronic low-grade inflammation in aging (Bruunsgaard, H. et al., Curr. Opin. Hematol., (2001), 8 (3):131-6), and atherosclerosis (Bochkov, V. N. et al., Nature, (2002), 419 (6902):77-81).